TNFa and IL-1p are proinflammatory mediators that induce a storm of chemokines and cytokines. They are thought to be responsible for prolonging production of inflammatory mediators in multiple chronic autoimmune and infectious diseases. We propose to investigate the mechanisms responsible for regulating responses to these prototype cytokines. Specifically, we will define the phosphatase requirements for controlling TNF and IL-1-mediated NF-KB activation. An RNAi approach will be used with a NF-KB-luciferase reporter. 500 siRNA constructs have been prepared and screened for basal and TNF-mediated signaling in SV40 large T antigen immortalized and primary cell cultures. Preliminary data demonstrate the effectiveness of this strategy. 19 phosphatase genes were identified in our initial screens. 10 of 19 genes were not previously associated with NF-KB signaling. The proposal lists three specific Aims. The first is to identify phosphatases involved in TNF and IL-1 signaling. This Aim details the methodology required to validate our initial findings, and applies our RNAi strategy to the study of IL-1 signaling. The function of each phosphatase gene associated with NF-KB signaling will be confirmed by over-expression. The second Aim investigates the direct and indirect targets for the phosphatases identified in Aim #1. We define points (kB degradation and nuclear translocation) in the signaling pathway above or below which the phosphatases are likely to operate. Co-immunoprecipitation of myc-tagged phosphatase components will be used to identify physical associations among NF-KB components and phosphatases. The subunits comprising the active PP1 and PP2A holoenzymes will be characterized. We also examine dephosphorylation of suspected target proteins, including TRAF2 and the p65 subunit of NF-KB. We then detail the mechanisms of action for a few phosphatases. The third Aim focuses on the ability of phosphatases to regulate transcription of endogenous genes. Preliminary data demonstrate the differential effects of phosphatase genes on regulation of chemokine and cytokine transcription. These studies will be extended to additional phosphatase genes, and we will examine transcriptional regulation of a selected series of NF-KB-dependent genes involved in the inflammatory process. In summary, the proposed experiments will provide insights into the role of phosphatases in controlling NF-KB-mediated signaling. We will also evaluate the impact of individual genes in controlling transcription of endogenous inflammatory mediators.